Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

13 p.-3 fig.-3 tab.A number of pharmaceutical steroid synthons are currently produced through the microbial side-chain cleavage of natural sterols as an alternative to multistep chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc2155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc2155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3-ketosteroid 9a-hydroxylase (KshAB) and a ketosteroid D1-dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, wehave constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4-androstadiene-3,17-dione. In addition,we have demonstrated that a double deletion mutant M. smegmatis MS6039-5941 [DMSMEG_6039 (DkshB1)and DMSMEG_5941 (DkstD1)] transforms natural sterols into 4-androstene-3,17-dione with high yields.These findings suggest that the catabolism of cholesterol in M. smegmatis mc2155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.This study was supported by grants from the Ministry of Science and Innovation (BFU2006-15214-C03-01, BFU2009-11545-C03-03) and Ministry of Economy and Competitiveness (BIO2012-39695-C02-01).Peer reviewe

Assembly in vitro of rhodococcus jostii RHA1 encapsulin and peroxidase DypB to form a nano-compartment

Rhodococcus jostii RHA1 peroxidase DypB has been recently identified as a bacterial lignin peroxidase. The dypB gene is co-transcribed with a gene encoding an encapsulin protein, shown in Thermotoga maritima to assemble to form a 60-subunit nano-compartment, and DypB contains a C-terminal sequence motif thought to target the protein to the encapsulin nanocompartment. R. jostii RHA1 encapsulin protein has been overexpressed in R. jostii RHA1, and purified as a high molecular weight assembly (Mr >106). The purified nanocompartment can be denatured to form a low molecular weight species by treatment at pH 3.0, and can be re-assembled to form the nanocompartment at pH 7.0. Recombinant DypB can be assembled in vitro with monomeric encapsulin to form an assembly of similar size and shape to the encapsulin-only nanocompartment, assessed by dynamic light scattering. The assembled complex shows enhanced lignin degradation activity per mg DypB present, compared with native DypB, using a nitrated lignin UV-vis assay method. The measured stoichiometry of 8.6 µmoles encapsulin/µmol DypB in the complex is comparable to the value of 10 predicted from the crystal structure

Obesidade infantil e a responsabilidade estatal com relação à publicidade ilícita de alimentos com baixo teor nutricional, à luz do princípio da proteção integral à criança e ao adolescente

Trabalho de Conclusão de Curso, apresentado para obtenção do grau de Bacharel no curso de Direito da Universidade do Extremo Sul Catarinense, UNESC.O presente estudo versa sobre a obesidade infantil como decorrência da exposição exagerada das crianças às publicidades de alimentos com baixo teor nutricional, as quais se aproveitam da hipossuficiência e vulnerabilidade das mesmas e utilizam-se de práticas enganosas e abusivas. Aborda, sobretudo, a responsabilidade do Estado diante desses atos, tendo em vista a violação ao princípio da proteção integral, bem como as possíveis ações estatais para garantir a defesa das crianças. Neste sentido, buscou-se esclarecer que, apesar de existirem legislações proibindo as publicidades abusivas e enganosas, as mesmas não são respeitadas na prática, ocasionando, como consequência, o descumprimento aos direitos inerentes à criança e ao adolescente. Para o desenvolvimento deste estudo adotou-se o método dedutivo, utilizando o procedimento monográfico com técnica de pesquisa bibliográfica

3-Ketosteroid 9 alpha-hydroxylase enzymes:Rieske non-heme monooxygenases essential for bacterial steroid degradation

Various micro-organisms are able to use sterols/steroids as carbon- and energy sources for growth. 3-Ketosteroid 9 alpha-hydroxylase (KSH), a two component Rieske non-heme monooxygenase comprised of the oxygenase KshA and the reductase KshB, is a key-enzyme in bacterial steroid degradation. It initiates opening of the steroid polycyclic ring structure. The enzyme has industrial relevance in the synthesis of pharmaceutical steroids. Deletion of KSH activity in sterol degrading bacteria results in blockage of steroid ring opening and is used to produce valuable C19-steroids such as 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione. Interestingly, KSH activity is essential for the pathogenicity of Mycobacterium tuberculosis. Detailed information about KSH thus is of medical relevance, and KSH inhibitory compounds may find application in combatting tuberculosis. In recent years, the 3D structure of the KshA protein of M. tuberculosis H37Rv has been elucidated and various studies report biochemical characteristics and possible physiological roles of KSH. The current knowledge is reviewed here and forms a solid basis for further studies on this highly interesting enzyme. Future work may result in the construction of KSH mutants capable of production of specific bioactive steroids. Furthermore, KSH provides an promising target for drugs against the pathogenic agent M. tuberculosis.</p

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_(cat)/K_m = 1000 ± 100 M^(−1) s^(−1) versus 700 ± 100 M^(−1) s^(−1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_(cat)/K_m = 80 ± 40 M^(−1) s^(−1)). In the presence of 3-HSA the K_(mapp) for O_2 was 100 ± 10 μM. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val^(367)–Val^(394)) could adopt two conformations differing by a rigid body rotation of 25° around Arg^(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids

A UTILIZAÇÃO DO FLUXO DE CAIXA NAS EMPRESAS: UM MODELO PARA A PEQUENA EMPRESA

Na atual conjuntura econômica há uma tendência natural no sentido deadotar-se a Demonstração de Fluxo de Caixa em substituição à Demonstraçãode Origens e Aplicações. Isto se deve fundamentalmente a sua menorcomplexidade o que facilita sobremaneira o entendimento, principalmente pelosusuários externos pouco habituados a termos técnicos.O fluxo de caixa nada mais é do que a distribuição no tempo de todas asentradas e saídas de numerário geradas pelas atividades da empresa, lidaportanto, com conceito de fácil assimilação, a disponibilidade financeira,facilitando o entendimento por todos.O escopo do presente artigo demonstra a importância da Demonstraçãodo Fluxo de Caixa quer seja apresentada juntamente com as demaisDemonstrações Contábeis ou isoladamente, especialmente no universo daspequenas e médias empresas, apresentando-se um modelo específico onde sepropõe que o mesmo seja utilizado como instrumento de controle gerencial